Formation of hairpins in situ using force-induced strand invasion

ABSTRACT

The present invention relates to a method of preparation of substrates for nucleic acid sequencing reactions. More specifically, the present invention provides a new method of preparing hairpins using force-induced strand invasion. Hairpins prepared by this method and methods of nucleic acid analysis using these hairpins are also part of the present invention.

The present invention relates to a method of preparation of substratesfor nucleic acid sequencing reactions. More specifically, the presentinvention provides a new method of preparing hairpins which can be usedin a single-molecule analysis process.

There are many situations when the quantity of nucleic acid (typicallyDNA or RNA) available to perform a genetic or epigenetic analysis islimited. Sample types include tumour biopsies, anthropology specimens,and forensic specimens. Amplification is usually performed to increasethe amount of starting material. Some major disadvantages, however, areassociated with amplification. In particular, the epigeneticmodifications are not conserved throughout the amplification process. Inaddition, amplification shows variable efficiency depending on thenumber and sequence of the different targets being simultaneouslyanalysed.

The inventors have previously designed a single-molecule analysis methodenabling the determination of a nucleic acid sequence (WO 2011/147931;WO 2011/147929), as well as a number of related applications, includingDNA detection and quantification (WO 2013/093005), and detection ofprotein binding to nucleic acids (WO 2014/114687). According to thismethod, a single nucleic acid hairpin is denatured by pulling on itsextremities e.g., with magnetic tweezers. Reducing the tension below athreshold allows for the renaturation of the hairpin. However, if thesaid denatured hairpin has been hybridised to a single-strandedoligonucleotide, then renaturation will be blocked, at leasttransiently. The sequence of the double-stranded molecule can then bedeciphered by determining the duration and/or location of the blockage.

This method requires the generation of hairpins which are attached atone end to a movable surface such as a bead, and at the other end toanother surface. Currently, DNA hairpins are produced using classic DNAlibrary construction techniques. Typically, such DNA hairpin structuresare obtained by taking double-stranded nucleic acid molecules ofinterest, and attaching synthetic oligonucleotides to each end, in orderto produce the required structure. This is typically performed using aDNA ligase enzyme. In addition to adding cost and time to the samplepreparation, the primary disadvantage of this approach for the purposesof the sequencing method devised by the inventors is that it results inthe production of a library of hairpins which must then be attached toparamagnetic beads (either during the reaction, or as a subsequentstep). Furthermore, in order to interrogate the molecules using amagnetic trap instrument, the beads must ultimately be fixed via thehairpin molecule to a surface (e.g. the floor of a flow cell).

In such a scheme, it then becomes critical to adjust the molar ratio ofhairpins to beads. If too many hairpins are used, the beads becomedensely covered with hairpins, and this can result in the attachment ofthe beads to the surface via multiple species of hairpins. This is ahighly undesirable situation, since the single-molecule analysis methodcan only be performed when each bead is attached to the surface via asingle hairpin, as more than one attachment point prevents the requiredfreedom of movement during the subsequent denaturing steps. Even forbeads that do become attached by one hairpin to the surface, any sampleDNA which is bound elsewhere on the bead will be lost to analysis, thussquandering valuable sample material. On the other hand, if too fewhairpins are used, the majority of beads will not bind to the surface atall as the probability of binding during the time which they are flowingthrough the flow-cell, is low.

Empirical results suggest that the optimal ratio of hairpins to beads inthe protocols discussed above is somewhere between approximately 100:1and 1000:1. While this is not a problem in situations where largequantities of the starting nucleic acids are available, in the sort ofscenarios where starting material is limited, it is critical to be ableto analyse a high percentage of the DNA molecules in a sample andwithout loss of quantitative or DNA modification information.

There is thus still a need for a method for preparing hairpins suitablefor single-molecule nucleic acid analysis while maintaining quantitativeaccuracy.

DETAILED DESCRIPTION

The inventors have now found a new method for constructing a hairpinstructure which can be used as a template for single-molecule analysis.Thus nucleic acid hairpins, suitable for subsequent analysis, can beassembled directly without the use of DNA enzymes (e.g. ligases), and insuch a way that the number of DNA molecules available for analysis ismaximised.

According to the method of the invention, hairpin assembly is brokeninto several discrete steps. The initial pre-hairpin production step isperformed using synthetic DNA components and, critically, withoutrequirement for the DNA sample of interest. It can thus be performed asa highly controlled process, in bulk, and long before the analysisitself is performed. Only the final hairpin construction requires theaddition of the nucleic acid of interest.

The present invention first provides a receiving nucleic acid molecule,already bound to two distinct surfaces, which can be manufactured andquality checked in advance. This is particularly advantageous in termsof costs and time. Receiving molecules of low quality can thus beidentified and discarded without wasting any precious molecules of thenucleic acid of interest, which was of course not the case with themethods of the prior art. The use of the input nucleic acid in themethod of the invention is optimal, resulting in minimal sample loss andan ability to work with low-concentration samples.

According to the present invention, a nucleic acid is formed, whereintwo polynucleotides are bound together through the hybridization of twocomplementary single-stranded regions A and A′ and each of thesepolynucleotides is bound to a distinct surface. In addition, at leastone of the two polynucleotides of this nucleic acid contains at leastone stretch of single-stranded nucleic acid C. This nucleic acid isdesignated HP1 (for “hairpin precursor 1”) and serves as a landing padfor the nucleic acid of interest. Such a structure can be formed easilyand reliably, by simply allowing the two complementary single-strandedregions A and A′ to hybridise.

According to the invention, the nucleic acid of interest is provided ina HP2 nucleic acid (“hairpin precursor 2”), wherein said nucleic acid ofinterest is a double stranded molecule bound at one end to a loop and atthe other end to the same two complementary regions A and A′ as in theHP1 structure. In addition, at least one of the two complementaryregions A and A′ is linked to a stretch of single-stranded nucleic acidC′, wherein C′ is complementary of C.

The inventors have surprisingly found that when said HP1 nucleic acid isdenatured, in presence of the HP2 molecule containing the nucleic acidof interest, an entirely new nucleic acid is formed, wherein C and C′hybridize, as well as A of HP1 with A′ of HP2, and A′ of HP1 with A ofHP2. The new nucleic acid molecule thus formed is a hairpin which isbound by its extremities to two different surfaces. One of saidextremities comprises A of HP1 hybridised with A′ of HP2, whereas theother extremity comprises the contiguous regions A′ and C of HP1respectively hybridised to the contiguous regions A and C′ of HP2. Thestem of the hairpin resulting from the process comprises the nucleicacid of interest. The hairpin thus formed behaves exactly like thehairpins constructed by the methods of the prior art. In particular, thehairpin of the invention is amenable to the same kind of single-moleculeanalysis as the hairpins of the prior art and under the same conditions(see e.g., WO 2011/147931; WO 2011/147929; WO 2013/093005; WO2014/114687).

In a first aspect, the invention thus relates to a method for preparinga hairpin, said method comprising the steps of:

-   -   a) providing a nucleic acid HP1, said nucleic acid comprising:        -   a first end bound to a first surface;        -   a single-stranded region of sequence A linked to said first            end,        -   a single-stranded region of sequence A′ hybridised to said            single-stranded region A, wherein said single-stranded            regions A and A′ are not covalently linked;        -   at least one single-stranded region of sequence C linked to            said single-stranded region of sequence A′;        -   A second end linked to said single-stranded region of            sequence C, wherein said second end is bound to a second            surface;    -   b) providing at least one nucleic acid HP2, said nucleic acid        comprising:        -   a double-stranded region comprising the sequence of            interest,        -   a loop linked to a first end of said double-stranded region,        -   a single-stranded region having the sequence A linked to a            first strand of the second end of said double-stranded            region, said region of sequence A being linked to a            single-stranded region of sequence C′, C′ being            complementary of C;        -   a single-stranded polynucleotide having the sequence A′            linked to a second strand of the second end of said            double-stranded polynucleotide molecule, wherein the            single-stranded polynucleotides of sequences A and A′ are            hybridised;    -   c) denaturing said nucleic acid HP1 of step c) in the presence        of said nucleic acid HP2 of step b); and    -   d) obtaining a hairpin.

As used herein, ‘hairpin’ means a nucleic acid molecule comprising aregion of intra-strand pairing linked to a loop. A ‘loop’, as usedherein, refers to a succession of nucleotides of a strand of saidnucleic acid that are not paired through hydrogen bonds with nucleotidesof the same or another strand of said nucleic acid. The region ofintra-strand pairing is usually referred to as a ‘stem’ and may compriseat least 1, preferably 2, more preferably 5, even more preferably 10,still more preferably 20 pairs of bases.

Preferably, obtaining a hairpin involves applying a physical force, suchas e.g., a tension, to HP1. More preferably, said tension is obtained bypulling away the ends of HP1.

In a preferred embodiment, the single-stranded region of sequence A ofsaid structure HP1 is linked to a single-stranded region of sequence D,said single-stranded region of sequence D being bound to said firstsurface. According to this embodiment, the single-stranded region ofsequence A′ of structure HP2 is linked to a single-stranded regionhaving the sequence D′, wherein D′ is complementary to D.

According to this embodiment, the hairpin formed comprises a firstextremity comprising the contiguous regions A and D hybridised to thecontiguous regions A′ and D′ of HP2, and a second extremity comprisingthe contiguous regions A′ and C of HP1 respectively hybridised to thecontiguous regions A and C′ of HP2.

Without being bound by theory, it can be hypothesized that the newhairpin structure is obtained by the formation of a Holliday junctionbetween HP1 and HP2, followed by the resolution of said junction. A“Holliday junction”, as used herein, refers to a four-way DNA junction,which is formed as a result of a reciprocal exchange of DNA strandsbetween two nearly identical DNA molecules. Holliday junctions aregenerally accepted to be the central intermediate in homologousrecombination. The resolution of these intermediates usually requiresvarious enzymes (see e.g., Matos & West, DNA Repair (Amst). 19: 176-181,2014). Importantly, no enzymatic activity is required for either formingor resolving the present Holliday junction. This is particularlyadvantageous, since it makes the process of forming the hairpin from theHP1 and HP2 nucleic acids irreversible as long as the tension is higherthan 1 pN. Furthermore, even in the absence of a tension exerted on thebead, it is possible to ensure that the strand invasion is irreversibleby creating a HP1 molecule comprising e.g. one or more mismatches and/ormodified bases in the sequences of A and A′. Such a mismatch or modifiedbases, though insufficient to significantly destabilize the binding ofthe two components of HP1, will yet create an energetic barrier thatwill prevent the strand invasion processes of proceeding in the reversedirection once it has moved past this region.

The first step in the formation of a Holliday junction is the invasionof a double-stranded nucleic acid molecule by a single strand. Theinventors have found that strand invasion of HP1 by HP2 is induced by apartial denaturation of HP1. By ‘denaturation’, it is herein meant theprocess of strands separation of a double-stranded nucleic acid moleculeoccurring when most of the hydrogen bonds between the said strands arebroken. The denaturation process yields a denatured nucleic acidmolecule, by which it is herein meant the two separated complementarystrands resulting from the denaturation of a double-stranded nucleicacid molecule. The denaturation may be partial, i.e., some of thehydrogen bonds between the two strands remain intact, or total, whereinall of said hydrogen bonds are broken. By ‘renaturation’, it is hereinreferred to the process by which two separated complementary strandsreform through hybridization into a double helix. As used herein,‘hybridization’ is the process of establishing a non-covalent,sequence-specific interaction between two or more complementary strandsof nucleic acids into a single hybrid.

There are several possibilities known to the skilled person to denaturea nucleic acid. In a most preferred manner, the two strands areseparated by submitting them to a physical force such as e.g., atension. For example, the free ends of the said double-stranded nucleicacid may be pulled apart, thus rupturing all the bonds between thepaired bases, and opening the double-stranded nucleic acid. Thisembodiment is particularly advantageous, as the inventors have foundthat exercising a small tension on the HP1 structure leads to a partialdenaturation thereof, thus inducing strand invasion by HP2.

A “nucleic acid” as used herein refers to a single- or double-strandedlinear polynucleotide containing either deoxyribonucleotides orribonucleotides that are linked by 3′-5′-phosphodiester bonds. Thenucleic acid of the invention can be in particular a DNA or an RNAmolecule, either natural or modified. The terms “deoxyribonucleic acid”and “DNA” as used herein mean a polymer composed ofdeoxyribonucleotides. The terms “ribonucleic acid” and “RNA” as usedherein mean a polymer composed of ribonucleotides. The saidsingle-stranded nucleic acid may also be made of modified nucleotides,such as 2,6-Diaminopurine (2-Amino-dA), 5-Methyl dC, locked nucleic acid(LNA), which are nucleotides in which the ribose moiety is modified withan extra bridge connecting the 2′ oxygen and 4′ carbon, or UnlockedNucleic Acids (UNAs) are acyclic RNA analogues without a C2′-C3′ bond inthe ribose ring. Said single-stranded nucleic acid can also comprisepeptide nucleic acid (PNA), wherein the backbone is composed ofrepeating N-(2-aminoethyl)-glycine units linked by peptide bonds.

A nucleic acid according to the invention may be double-stranded and/orsingle-stranded. A double-stranded nucleic acid comprises two strandspaired in anti-parallel orientation through hydrogen bonds, wherein thebase sequence on one strand is complementary of the base sequence on theother strand. Most often, the double-stranded nucleic acid will be DNA,but it is understood that the invention also applies to single-strandedDNA-single-stranded DNA duplexes, perfectly paired or not perfectlypaired, or alternatively to single-stranded DNA-single-stranded RNAduplexes, perfectly paired or not perfectly paired, or alternatively tosingle-stranded RNA-single-stranded RNA duplexes, perfectly paired ornot perfectly paired. Furthermore, the duplex may consist of the atleast partial re-pairing of two single strands obtained from samples ofdifferent origins. Finally, the invention also applies to the secondarystructures of a sole single-stranded DNA or of a sole single-strandedRNA. The single-stranded nucleic acid of the invention can be inparticular a DNA or an RNA molecule, either natural or modified.

In a preferred embodiment, the nucleic acid of the invention containsboth regions which are double-stranded and regions which aresingle-stranded. In other words, in this embodiment, some regions of thenucleic acid are specifically paired to their complementary sequence ina duplex, while other regions are not hybridised. Such single-strandregions are thus free to hybridize with polynucleotides possessingcomplementary regions. In particular, the single-stranded region(s) ofthe nucleic acid HP2 is (are) free to hybridize with the complementaryregion(s) present in HP1, provided HP1 is partially denatured.

In a typical configuration, the nucleic acid HP1 is specificallyanchored on two solid surfaces (e.g. microscope slide, micropipette,microparticle), one of which can be moved. In other words, one of theends is attached directly or indirectly to a surface, while the otherend is attached directly or indirectly to a movable surface. In thisembodiment, a tension is applied on both ends of the HP1 molecule whenthe surfaces are moved away. When the tension is higher than a thresholdvalue, the two strands of the double-stranded regions of HP1 areseparated and the nucleic acid molecule is at least partially denatured,thus enabling strand invasion by the single-stranded regions of HP2. Thetension applied is preferentially above or equal to 3 pN; it is morepreferentially above or equal to 4 pN; it is even more preferentiallyabove or equal to 5 pN; in a very much preferred aspect, it is above orequal to 6 pN. In a preferred embodiment, the tension applied is notsufficient for fully denaturing HP1. According to this embodiment, thetension applied is preferentially below or equal to 11 pN; it is morepreferentially below or equal to 10 pN; it is even more preferentiallybelow or equal to 9 pN; in a very much preferred aspect, it is below orequal to 8 pN. This force may vary with temperature, nucleotide type andbuffer, but the skilled person will easily adapt the said force withregard to these parameters in order to obtain the partial separation ofthe two strands.

In a preferred embodiment, HP1 nucleic acid molecules are anchored atmultiple points at one end to a motionless element, e.g. a surface, andat the other end to a movable surface, in this case a magnetic bead.Magnets are provided for acting on the bead. In particular, the magnetsmay be used for pulling the bead away from the surface. However, theimplementation of the method of the invention is not restricted to theabove apparatus. Any device which allows one to fully extend and thenrefold a molecule of double stranded nucleic acid, whilst monitoring atthe same time the extension of the said molecule can be used toimplement the method of the invention. For example, acoustic or opticaltweezers may be used; they require however prior force calibration andare not easily parallelized for high throughput measurements. Furtherdrawbacks of the optical tweezers are the lack of total torsionalcontrol of the nucleic acid and the possible local heating of thesolution by the focussed laser which may alter the hybridizationconditions.

It will be immediately apparent to the skilled person that the moleculesof HP1 can be prepared in advance. For example, in a first step, a firstnucleic acid molecule comprising a single-stranded region of sequence Aand a second nucleic acid molecule comprising a single-stranded regionof sequence A′ and at least one single-stranded region of sequence Clinked to said single-stranded region of sequence A′, wherein A, A′ andC are as defined above, can be prepared by conventional molecularbiology techniques. The complementary regions A and A′ are then allowedto hybridize. In another step, the two ends of HP1 are bound to twosurfaces, on which can be moved (e.g., with acoustic, optical, ormagnetic tweezers). This binding step and the hybridization step can beperformed in any order, although it is preferred that the sequences Aand A′ have been allowed to form a duplex before the ends of the nucleicacid molecule are bound to the surfaces. The sequences A and A′ can beof any length, provided they are long enough to maintain the twopolynucleotides of HP1 together, without requiring the formation of acovalent bond. Preferably, each of said sequences comprises at least 30,more preferably 35, even more preferably 40, still more preferably 45nucleotides. The combined regions A′ and C and/or the combined regions Aand D should be long enough for resisting the maximum shearing forceproduced by pulling on the ends of the hairpin. In particular, each ofthe sequences C and C′ comprises at least 10, more preferably 12, evenmore preferably 13, still more preferably 14, most preferably 15nucleotides. Likewise, the sequence D and the sequence D′ each comprisesat least 8, more preferably 10, even more preferably 11, still morepreferably 12, most preferably 13 nucleotides.

The nucleic acid HP1 is incubated for a few minutes in a solution ofadequate beads (for example streptavidin coated ones) to which it bindsby one of its labelled (for example biotin) ends. The beads can betransparent if optical tweezers are later used for manipulation ormagnetic if one uses magnetic traps or tweezers for manipulation. Thereis no restriction with regard to the nature of the beads for usingacoustic beads.

The bead-nucleic acid assembly is injected in a fluidic chamber thesurface of which has been treated such as to bind the other labelled endof the molecule (for example a surface coated with anti-Dig antibodiesto bind the Dig-labelled end of the nucleic acid). The beads are thusanchored to the surface via the molecules of HP1. The distance of thebead to the surface is then monitored by various means known to the manof the art: for example the diffraction rings of their image on a cameracan be used to deduce their distance, or the light intensity theyscatter (or emit by fluorescence) when illuminated in an evanescent modecan be used to measure their distance. Alternatively, the magnetic fieldthey generate can be measured (using a magnetic sensor such as GMR orHall sensors) to deduce their distance to a sensor on the anchoringsurface.

To pull on the nucleic acid molecule anchoring the beads to the surfacevarious techniques have been described. One can use the light of afocused laser beam to trap a transparent bead near the focal point. Bythe relative translation of the beam with respect to the anchoringsurface one can apply a force on the tethering molecule (a typicaloptical tweezers assay). The exerted force being proportional to thedisplacement of the bead from its equilibrium position, to exert aconstant force on the tethering molecule requires a feedback loop on thetrapping beam.

To exert a constant force on a bead, the use of the hydrodynamic draggenerated by a flow around the bead has been described, but it usuallyyields a low spatial accuracy (>100 nm). The preferred embodiment uses amagnetic trap to pull on super-paramagnetic beads anchored to a surfaceby a nucleic acid hairpin as described above. In this configuration,small magnets placed above the sample are used to apply a constant forceon the anchored bead, whose position can be determined with <1 nmaccuracy (depending on the pulling force and the dissipation due tohydrodynamic drag)

In order to attach nucleic acids to surfaces or supports, use may bemade of any one of the techniques known in the field. Essentially, thenucleic acid becomes anchored directly to the support, for example themicro-bead, which involves a functionalization of this surface, forexample by coating it with streptavidin, a COOH group, and the like,capable of reacting with the functionalized end of the nucleic acid.

Such methods necessitate, in general, functionalizing the nucleic acid,especially the 3′ and 5′ ends, that is to say grafting appropriatechemical groups onto them. For this purpose, different procedures may beadopted. The simplest is to functionalize, using syntheticoligonucleotides, each of the ends of a HP1 with two differentfunctions, which permit anchoring to two different pre-treated surfaces.This enables the two ends to be treated differently. For example, afirst synthetic oligonucleotide containing a biotin at its 5′ end may beused to obtain a first adapter, which is then linked to a first end ofthe HP1 molecule, thus enabling coupling to a streptavidin-coatedsurface. Likewise, a second synthetic oligonucleotide may be used toobtain a second adapter, said adapter containing Digoxigenin-labellednucleotides. This second adapter may then be linked to a second end ofthe HP1 molecule, thus enabling coupling to an anti-Dig-antibody-coupledsurface. Advantageously, said anti-Dig-antibody-coupled surface isdifferent from the streptavidin-coated surface. Preferably, the bead hasbeen coated with streptavidin, while said anti-Dig-antibody-coupledsurface is the surface of the fluidic chamber into which thebead-nucleic acid complex is injected. The resulting HP1 molecules arethus anchored through a first end to the bead and through the other endto the flow cell. The advantage of this method lies in its capacity toseparately functionalize a heterogeneous population of large nucleicacid fragments (as are obtained by fractionation of a gene orchromosome), during the production of a library of HP2 precursors. Thesecan then be analysed simultaneously.

The drawback of this method lies in the steric interference between thetwo adjacent functional groups, which can make coupling to the surfacesdifficult. To solve this problem, it can be advantageous to add at eachfree end of the hairpin molecule a “spacer” sequence of bases, to theend of which a functional group is then added; the two spacer sequencesare non-complementary, affording each functional group enough space tobind to its dedicated surface. In addition, said spacers serve to keepthe nucleic acid further away from the surface of the bead or flow cell,and thus minimize electrostatic repulsion. More advantageously, thesequence of each spacer sequence is designed in order to usesingle-stranded sequencing primers of known sequence in the sequencingmethod of the invention. These spacers can be single stranded or doublestranded or a mixture of both. Double stranded spacers are preferredsince they are more rigid, which helps to keeps the hairpin away fromthe surfaces. Moreover, if a nick (i.e., a break in the phosphodiesterbackbone) appears in a long double-stranded nucleic acid, thefunctionality of the hairpin may be preserved, i.e., such a nickedhairpin may still be used for analytic purposes in the methodspreviously developed by the inventors (see e.g., WO 2011/147931; WO2011/147929; WO 2013/093005; WO 2014/114687).

Methods for preparing such spacers and adding them to HP1 are well knownto the person skilled in the art and need thus not be detailed here.

As regards the actual anchoring techniques, there are many of these andthey derive from the techniques for anchoring macromolecules (proteins,DNA, and the like) to commercially available pretreated surfaces. Mostof these techniques have been developed for immunology tests, and linkproteins (immunoglobulins) to surfaces carrying groups (—COOH, —NH₂,—OH, and the like) capable of reacting with the carboxyl (—COOH) oramine (—NH₂) ends of proteins.

The covalent anchoring of nucleic acid may be accomplished directly, viathe free phosphate of the 5′ end of the molecule, which reacts with asecondary amine (Covalink —NH surface marketed by Polylabo atStrasbourg) to form a covalent bond. It is also possible tofunctionalize DNA with an amine group and then to proceed as with aprotein.

There are also surfaces coated with streptavidin (Dynal beads, and thelike), which permit quasi-covalent anchoring between the streptavidinand a biotinylated DNA molecule. Lastly, by grafting an antibodydirected against digoxigenin onto a surface (by the methods mentionedabove), a nucleic acid functionalized with digoxigenin may be anchoredthereto. This represents merely a sample of the many possible anchoringtechniques (see e.g., Janissen et al., Nucleic Acids Res. 42(18):e13,2014).

Among the attachment and anchoring techniques, there should also bementioned, for example, the techniques described in Patent EP 152 886using an enzymatic coupling for the attachment of DNA to a solid supportsuch as cellulose.

Patent EP 146 815 also describes various methods of attachment of DNA toa support.

Similarly, patent application WO 92/16659 proposes a method using apolymer to attach DNA.

Naturally, the nucleic acid may be attached directly to the support but,where necessary, especially with a view to limiting the influence of thesurfaces, the nucleic acid may be attached at the end of an inert arm ofpeptide or other nature, as is, for example, described in Patent EP 329198.

Substrates or supports for use in the invention include, but are notlimited to, latex beads, dextran beads, polystyrene surfaces,polypropylene surfaces, polyacrylamide gel, gold surfaces, glasssurfaces and silicon wafers. In certain embodiments, the solid supportmay include an inert substrate or matrix that has been functionalized,for example by the application of a layer or coating of an intermediatematerial including reactive groups that permit covalent attachment tomolecules such as polynucleotides.

In a preferred embodiment, the substrate for use in the inventionenables the attachment of several independent HP1 molecules at discretelocations, so as to allow the simultaneous construction of a greatnumber of hairpins by the method of the invention, thus enabling thesimultaneous analysis of a great number of nucleic acid molecules. Thus,according to this embodiment, more than one HP1 molecules, for example,at least two or three or four or more, HP1 molecules may be grafted tothe solid support. For example, the HP1 molecule is advantageously boundto the surface of each of the wells of a microarray. In this manner, alibrary of nucleic acid sequences of interest can be utilized in themethod of the invention with the HP1 molecules bound to the surface toprepare a library of hairpins. This library of hairpins, wherein eachhairpin contains a specific nucleic acid sequence, is suitable for usein applications usually carried out on ordered arrays such asmicro-arrays. Such applications by way of non-limiting example includehybridization analysis, gene expression analysis, protein bindinganalysis, sequencing, genotyping, nucleic acid methylation analysis andthe like (WO 2011/147931; WO 2011/147929; WO 2013/093005; WO2014/114687). The clustered array may be sequenced before being used fordownstream applications such as, for example, hybridization with RNA orbinding studies using proteins.

Common molecular biology techniques can be used to prepare the moleculesof HP2 by ligating a loop to the nucleic acid of interest. Likewise, theother extremity of the nucleic acid of interest may be ligated to theregions A and A′ using only usual methods of molecular biology. Forexample, an oligonucleotide comprising the regions A and C′ may besynthesized and hybridised to another oligonucleotide, said otheroligonucleotide having the sequence A′. This results in adouble-stranded polynucleotide with a single-stranded overhang. Saidpolynucleotide can then be ligated to the nucleic acid of interest.

The nucleic acid of interest according to the invention may be any typeof nucleic acid. The nucleic acid of interest can be synthetic orderived from naturally occurring sources, or may include both syntheticand natural sequence; and may include PCR products. In this particularembodiment, said nucleic acid of interest is a single species of nucleicacid, i.e., all the molecules of said nucleic acid are identical. Inthis case, all the molecules of HP2 will be identical and therefore allthe hairpins prepared by the method of the invention will likewise beidentical.

In another embodiment, the nucleic acid of interest represents apopulation of double-stranded nucleic acid molecules. This is the case,for example, when a library of nucleic acid sequences, such as e.g., agenomic library or an expression library is used to prepare the HP2molecules. This results in a population of HP2 molecules wherein eachmolecule is distinct from the other, thus generating with the method ofthe invention a population of unique hairpins which can be directlyanalysed or sequenced by the methods devised by the inventors (WO2011/147931; WO 2011/147929; WO 2013/093005; WO 2014/114687). Accordingto this embodiment, the molecules of the nucleic acid of interest arefor example isolated from a biological sample containing a variety ofother components, such as proteins, lipids and non-template nucleicacids. A “biological sample” may be any sample which may contain abiological organism, such as, for example, bacteria, viruses, plants,yeasts etc. A “biological sample” according to the invention also refersto a sample which may be obtained from a biological organism, such as acellular extract obtained from bacteria, viruses, plants, yeasts etc.Molecules of the nucleic acid of interest can be obtained directly froman organism or from a biological sample obtained from an organism, e.g.,from blood, urine, cerebrospinal fluid, seminal fluid, saliva, sputum,stool and tissue. Any tissue or body fluid specimen may be used as asource for nucleic acid for use in the invention. Molecules of thenucleic acid of interest can also be isolated from cultured cells, suchas a primary cell culture or a cell line. The cells or tissues fromwhich template nucleic acids are obtained can be infected with a virusor other intracellular pathogen. A sample can also be total RNAextracted from a biological specimen, a cDNA library, viral, or genomicDNA. Nucleic acid obtained from biological samples typically isfragmented to produce suitable fragments for analysis. In oneembodiment, nucleic acid from a biological sample is fragmented bysonication. Molecules of the nucleic acid of interest can be obtained asdescribed in US 2002/0190663. Generally, nucleic acid can be extractedfrom a biological sample by a variety of techniques such as thosedescribed by Maniatis, et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor, N.Y., pp. 280-281 (1982). Generally, individualnucleic acid template molecules can be from about 1 base to about 20 kb.These individual molecules are then used to prepare the HP2 molecules asdescribed above. Thus in this embodiment, the method of the inventioncomprises using a plurality of distinct HP2.

In another aspect, the present invention provides a HP1 and a HP2molecule for use in the method described above.

The present invention also provides a hairpin obtained by the method ofthe invention. Said hairpin comprises:

-   -   A first end bound to a first surface,    -   A first double-stranded region linked to said first end, wherein        said first double-stranded region comprises a first strand        comprising a region A and a second strand comprising a region        A′, wherein A and A′ are hybridised;    -   A stem-and-loop region linked to said first double-stranded        region, wherein said stem comprises the nucleic acid of        interest;    -   A second double-stranded region linked to said stem-and-loop        region, wherein said second double-stranded region comprises a        first strand comprising a region A and a region C′ and a second        strand comprising a region A′ and a region C, wherein the        regions A and C′ are hybridized with the regions A′ and C,        respectively;    -   A second end linked to said second double-stranded region,        wherein said second end is bound to a second surface.

In a preferred embodiment, said first double-stranded region comprises afirst strand comprising a region A and a region D and a second strandcomprising a region A′ and a region D′, wherein the regions A and D arehybridised with the regions A′ and D′, respectively

In another preferred embodiment, one of the surfaces is a movablesurface, as described above.

The hairpin of the invention comprises a double-stranded stem and anunpaired single-stranded loop. In a hairpin, the ends of the two strandswhich are not engaged in the loop are free and can thus be pulled apart.This results in the unpairing of the double stranded nucleic acid stem,thus yielding a denatured double stranded nucleic acid molecule. It ispossible to open completely a hairpin double-stranded nucleic acidmolecule by pulling on each end of the said nucleic acid molecule with aforce higher than a threshold value. When the tension applied to themolecule is decreased to an intermediate value, the nucleic acidmolecule self-rehybridises to reform a hairpin.

It is advantageous in this respect to design the loop sequence andlength so that the hairpin refolds after a short transient, e.g. 1 s.Methods to this effect have been described in the prior art, e.g. inWoodside et al., Proc. Natl. Acad. Sci. U.S.A., 103 (16): 6190-6195,2006). When the force is decreased from the opening to the test value,the extension of the open hairpin varies because of the elasticity ofsingle stranded DNA. The small delay before the hairpin refolds allowsthe user to determine the hairpin extension at the same force as the oneused to detect the blocking state.

The hairpin formed by the method of the invention can be denatured andrenatured, as usual for hairpin structures, and is suitable for allmapping and sequencing experiments. For example, if one of the twosurfaces is a movable surface, pulling said movable surface away fromthe other generates a tension which results in a hairpin molecule whichis denatured at least partially. In fact, the inventors havesurprisingly found that the length of the complementary sequence(AC/A′C′, and A′/A; or A′C/AC′ and A/A′) is long enough to resist theshearing force of the movement of the surface, as opposed to the smallerlength of complementary sequence found in the Holliday junction.

Therefore, in yet another aspect, the invention relates to a method ofanalysis of the nucleic acid of interest comprised in the hairpinobtained by any of the method described above.

For example, the hairpin can be used for detecting the nucleic acid ofinterest. According to this embodiment, when a single-stranded nucleicacid molecule is added to a denatured double-stranded nucleic acid priorto renaturation, a blockage of rehybridization indicates that thesequence of the single-stranded nucleic acid molecule is complementaryto at least part of the sequence of the double-stranded nucleic acidmolecule.

This single-stranded nucleic acid can be of any length, provided that itis long enough to block the renaturation process. Preferentially, thelength of the single stranded nucleic acid will be comprised between 3and 50 nucleotides; more preferentially, between 3 and 45 nucleotides,between 3 and 40 nucleotides, between 3 and 35 nucleotides, between 3and 30 nucleotides, between 3 and 25 nucleotides, between 3 and 20nucleotides, between 3 and 15 and even more preferentially between 3 and12. The single-stranded nucleic acid of the invention can be inparticular a DNA or an RNA molecule, either natural or modified. Saidsingle-stranded nucleic acid may also be made of modified nucleotides,such as 2,6-Diaminopurine (2-Amino-dA), 5-Methyl dC, locked nucleic acid(LNA), which are nucleotides in which the ribose moiety is modified withan extra bridge connecting the 2′ oxygen and 4′ carbon, or UnlockedNucleic Acids (UNAs) are acyclic RNA analogues without a C2′-C3′ bond inthe ribose ring. Said single-stranded nucleic acid can also comprisepeptide nucleic acid (PNA), wherein the backbone is composed ofrepeating N-(2-aminoethyl)-glycine units linked by peptide bonds. Inanother embodiment, the said single-stranded nucleic acid may alsocontain a modification at the end of the oligo (5′ or 3′) that improvesbinding. Well known examples of such modifications include the MGB(Minor Groove Binder), acridine (an intercalator) and ZNA (i.e., zipDNA, a spermine derivative).

Thus, the method of the invention also relates to a method for detectingsaid nucleic acid sequence, said method comprising the steps of:

-   -   a) denaturing a hairpin molecule as described above, said        hairpin potentially comprising said nucleic acid sequence, by        applying a physical force to said hairpin molecule;    -   b) providing a single-stranded nucleic acid molecule;    -   c) renaturing the said hairpin molecule in the presence of the        said single-stranded nucleic acid molecule    -   d) detecting a blockage of the renaturation of the hairpin.

Preferably, the physical force of step a) is a tension, which is appliedby moving away the supports. When the tension is higher than a thresholdvalue, the two strands are separated and the nucleic acid molecule isdenatured. The tension applied is preferentially above or equal to 15pN; it is more preferentially above or equal to 16 pN; it is even morepreferentially above or equal to 17 pN; in a very much preferred aspect,it is above or equal to 18 pN. This force may vary with temperature,nucleotide type and buffer, but the skilled person will easily adapt thesaid force with regard to these parameters in order to obtain theseparation of the two strands. On the other hand, when the tension isdecreased under a minimal value, the two strands of the denatureddouble-stranded nucleic acid can rehybridise. To obtain rehybridizationof the said two strands, a tension of less than or equal to 12 pN ispreferentially applied; more preferentially, it is less than or equal to11 pN; even more preferentially, it is less than or equal to 10 pN.Preferably, said tension is higher than 1 pN, to prevent reversibilityof strand invasion. Alternatively, the tension may be as low as 0 pNwhen the HP1 molecule comprises one or more mismatches and/or modifiedbases can be introduced in the sequences of A and A′, which isinsufficient to significantly destabilize the binding of the twocomponents of HP1, and yet will create an energetic barrier that willprevent the strand invasion processes of proceeding in the reversibledirection once it has moved past this region.

Using a hairpin makes it possible, in particular, to perform cycles ofpairing and unpairing and thus to improve the signal/noise ratio.

By determination of the blockage, it is herein meant the determinationof the physical parameters associated with the blockage. The most usefulof these parameters is the position of the blockage on thedouble-stranded nucleic acid molecule, said position corresponding tothe position of hybridization of the single-stranded nucleic acidmolecule on the double-stranded nucleic acid molecule. Indeed, theinventors have found that the position on the double-stranded nucleicacid at which the pause in renaturation occurs can be preciselydetermined: the use of a hairpin affords the skilled person a means todetermine the physical distance between the two free ends of the hairpinat any time during the denaturation/renaturation process.

By ‘free end’ it is herein meant the end of one strand which is notcovalently linked to an extremity of the other strand; as explainedabove, these free ends are each bound to a different surface. Inparticular, one of these surfaces is movable, whilst the other may bemotionless. The skilled person will thus easily realize that, in orderto measure the distance between the free ends of the hairpin, it ispossible to simply measure the distance between the two surfaces.

This distance is maximal (z_(high) (F_(open))) when the hairpin moleculeis completely denatured, since the hairpin nucleic acid is thencompletely extended; it is minimal (z_(low) (F_(test))) when the saidhairpin molecule is completely renatured. It is advantageous to performall length comparisons at the same force F_(test), so that the singlestranded nucleic acid has the same elastic properties. Using the delayin loop closing the skilled user can measure z_(high) (F_(test)).Likewise, the distance between the two free ends when the renaturationprocess is temporarily paused can be measured: as expected, thisdistance z is comprised between t_(high) and z_(low) (all z beingmeasured with F=F_(test)). It is immediately clear that the distance zvaries with the localization in the hairpin molecule of the sequence towhich the sequence of the single-stranded nucleic acid is complementary.If the said single-stranded nucleic acid hybridises with a sequencewhich is located close to the free ends of the hairpin, theself-rehybridization process is blocked just before the complete hairpinis reformed; in this case, z_(pause) is minimal. On the other hand, ifthe said single-stranded nucleic acid hybridises with a part of thehairpin which is close to the unpaired loop, the renaturation processwill be arrested in a situation where the hairpin is completely, oralmost completely denatured; in this case, z_(pause) is maximal.

It is possible to correlate precisely a physical distance in adouble-stranded nucleic acid molecule with a number of bases. Forexample, a distance of 1 nm corresponds to the distance spanned by twonucleotides (1 bp) in a nucleic acid under a 10 pN force. The exactcalibration versus force is given by the elasticity of single strandednucleic acid. Therefore, by simply measuring the distance between thetwo free ends of the hairpin molecule, it is possible to determineprecisely where the renaturation is blocked.

In a preferred embodiment, the detection of the blockage of therenaturation of the said hairpin involves determining the position ofthe blockage on the hairpin, as described above.

Thus, in one embodiment, the invention consists of a method fordetermining the sequence of a nucleic acid, wherein the hairpin moleculecomprising the sequence to be determined is first denatured byapplication of a physical force, then rehybridised in a presence of asingle-stranded nucleic acid, and the presence of a blockage in therehybridization detected. In one aspect, the distance between the twoends of the hairpin is determined when the renaturation process isblocked. Preferentially, the distance between the two ends of saidhairpin is determined when the molecule is completely denatured. Evenmore preferentially, the two distances are compared and the position ofthe blockage is determined.

According to this particular embodiment, the method for determining thesequence of a nucleic acid comprises the steps of:

-   -   a) denaturing a hairpin obtained by the method described above,        said hairpin comprising said nucleic acid sequence, by moving        away the surfaces;    -   b) measuring the distance Z_(high) between the two ends of the        denatured hairpin molecule obtained in step a);    -   c) hybridizing a single-stranded nucleic acid molecule, the        primer, with said denatured hairpin molecule obtained in step        a);    -   d) renaturing said hybridised single-stranded nucleic acid        molecule/hairpin molecule of step c); and    -   e) detecting a blockage of the renaturation of the hybridised        single-stranded nucleic acid molecule/hairpin molecule; and    -   f) determining the position of said blockage with respect to one        end of the hairpin, said determination comprising the steps of:        -   measuring distance (z) between the two ends of the hairpin            molecule which are attached to a support,        -   comparing z and Z_(high), and        -   determining the position of the blockage;            wherein said nucleic acid sequence is derived from the            position of said blockage.

In a particular embodiment, the sequencing of the nucleic acid ofinterest contained in the hairpin molecule of the invention involves thereplication of said nucleic acid with a polymerase. For example, thepolymerase reaction may be performed in presence of a pool ofdeoxy-nucleotides (dNTP) where one of the bases is present at a very lowconcentration. In that case, each time the polymerase encounters thecomplement of the said nucleotide, it pauses until the low concentrationnucleotide diffuses into position (Greenleaf and Block, Science, 313:801, 2006; U.S. Pat. No. 7,556,922). Alternatively, it is possible touse dideoxynucleotides (ddNTPs) in addition to the normaldeoxynucleotides (dNTPs) found in DNA. Incorporation of one ddNTP causesthe polymerase reaction to stop, since no nucleotide can be added afterthe said ddNTP. The position of each pause or blockage can then bedetermined by the method of the invention, i.e. by measuring thephysical distance between the two free ends of the molecule, thusleading to the identification of the sequence of the nucleic acidcontained in the hairpin of the invention. Further embodiments of thismethod can be found in WO 2011/147929.

Another useful parameter associated with the blockage in renaturation isthe period of time during which the renaturation is blocked (referredherein as the duration of the pause in renaturation). Indeed, it ispossible to measure the period of time during which the rehybridizationis blocked. For example, the skilled person can determine the period oftime during which the distance between the two ends of the hairpin is zas defined above, i.e. an intermediate value comprised between Z_(high)and Z_(low).

Thus, in one particular embodiment, the method for determining thesequence of a nucleic acid comprises the steps of:

-   -   denaturing the said hairpin molecule comprising said nucleic        acid sequence by applying a physical force to said hairpin;    -   providing a single-stranded nucleic acid molecule,    -   renaturing the hairpin molecule in the presence of the said        single-stranded nucleic acid molecule; and    -   detecting a blockage of the renaturation of said hairpin, and    -   determining the duration of said blockage.

The duration of the blockage is dependent upon the degree ofcomplementarity between the two sequences. The higher thecomplementarity, the greater the number of bonds established between thetwo molecules, and therefore the longer the duration.

In this particular embodiment, the method according to the presentinvention may thus be used for diagnostic purposes. In particular, it ispossible to provide for a simplified technique, based on the observationthat a mismatch between the single-stranded nucleic acid and thesequence of interest results in a much shorter lived hybridization. In afirst aspect, the renaturation of a hairpin is blocked by asingle-stranded nucleic acid, by any of the methods described above, andthe duration of the blockage is determined. In a preferred aspect, thisvalue is compared to a reference value. In a further preferred aspect,the reference value corresponds to the length of the pause observed witha reference single-stranded nucleic acid, as determined by any of theabove methods.

In this embodiment, the invention relates to a method for detecting thepresence of a specific nucleic acid sequence, said method comprising thesteps of:

-   -   a) denaturing a hairpin obtained by the method described above,        said hairpin potentially comprising said sequence, by moving        away the surfaces;    -   b) measuring the distance Z_(high) between the two ends of the        denatured hairpin molecule obtained in step a);    -   c) hybridizing a single-stranded nucleic acid molecule, the        primer, with said denatured hairpin molecule obtained in step        a);    -   d) renaturing said hybridised single-stranded nucleic acid        molecule/hairpin molecule of step c); and    -   e) detecting a blockage of the renaturation of the hybridised        single-stranded nucleic acid molecule/hairpin molecule; and    -   f) determining the position of said blockage with respect to one        end of the hairpin, said determination comprising the steps of:        -   measuring distance (z) between the two ends of the hairpin            molecule which are attached to a support,        -   comparing z and Z_(high), and        -   determining the position of the blockage;    -   g) determining the duration of said blockage,        wherein the presence of said nucleic acid sequence is derived        from the position of said blockage and the duration of said        blockage.

This method is particularly useful since it enables the detection of onesingle molecule within a whole population of nucleic acid molecules.Because of the single-molecule resolution obtainable with the method ofthe invention, each molecule carrying a specific sequence can bedetected. Thus the present invention affords the skilled person tonumerate the number of nucleic acid molecules carrying the saidsequence. The present method allows for the easy and accuratequantification of a specific nucleic acid sequence in a whole populationof nucleic acid molecules.

The method of the invention is particularly suited for generating alibrary of hairpins, each hairpin comprising a specific nucleic acidmolecule. This method is thus particularly convenient for detecting asequence of interest, e.g. a particular allele, within a wholepopulation of nucleic acid molecules, for example in a biologicalsample. In this embodiment, a library of hairpins is obtained by themethod described above, wherein said hairpin library represent a wholepopulation of nucleic acids. Each of the hairpins of said library isthen denatured by applying a tension to the ends of said hairpins, e.g.,by moving away the surfaces. A single-stranded nucleic moleculecomprising the sequence of interest is provided and allowed to hybridiseto the denatured hairpin molecules. The hairpins are then renatured inthe presence of said single-stranded nucleic acid molecule by e.g.,reducing the tension applied to the ends. A pause in the renaturation ofthe hairpin is detected, and the duration of said pause is determined.According to this embodiment, the longest pause will be observed for thehairpin(s) comprising a nucleic acid whose sequence is exactlycomplementary of the sequence of interest. Because of thesingle-molecule resolution obtainable with the method of the invention,each molecule carrying a specific sequence can thus be detected. Thusthe present invention affords the skilled person to numerate the numberof nucleic acid molecules carrying the said sequence. Furtherembodiments and applications of the present method for the easy andaccurate quantification of a specific nucleic acid sequence in a wholepopulation of nucleic acid molecules can be found in WO 2013/093005.

In another embodiment, the hairpins of the invention are used fordetecting the binding of a protein to a specific sequence. According tothis embodiment, the invention relates to a method for the determinationof the binding of a protein to a sequence of interest, said sequencebeing contained within a hairpin obtained by the method described above,wherein said method comprises a step of blocking the renaturation ofsaid hairpin. More specifically, the hairpin is first denatured byapplying a physical force, such as e.g., a tension, to said molecule(e.g., by moving away the surfaces to which the ends of said hairpin arebound). The protein is then provided and, optionally, a single-strandednucleic acid molecule corresponding to said sequence. Said protein isallowed to bind to the sequence of interest (either as a denaturedsingle-stranded hairpin, or a as a duplex between said denatured hairpinand said single-stranded nucleic acid molecule), before the hairpin isrenatured by reducing the tension. A pause in the renaturation of thehairpin is detected, and the localization of said pause is determined asindicated above. Optionally, the duration of the pause is alsodetermined.

The terms ‘protein’, ‘proteins’, ‘polypeptide’, and ‘polypeptides’, asused herein, are synonyms and refer to polymers of amino acidscovalently linked through peptide bonds into a chain. Proteins can haveseveral functions. A ‘binding protein’ is a protein which is capable ofbinding non-covalently to another molecule. A binding protein can bindto, for example, a DNA molecule (a DNA-binding protein), an RNA molecule(an RNA-binding protein) and/or a protein molecule (a protein-bindingprotein). In the case of a protein-binding protein, it can bind toitself (to form multimers) and/or it can bind to one or more moleculesof a different protein or proteins. A binding protein can have more thanone type of binding activity. For example, zinc finger proteins haveDNA-binding, RNA-binding and protein-binding activity. A ‘nucleicacid-binding protein’ according to the invention is thus a protein whichis capable of interacting with a nucleic acid. A ‘single-strandednucleic acid-binding protein’ according to the invention is thus aprotein which is capable of interacting with a single-stranded nucleicacid, while a ‘double-stranded nucleic acid-binding protein’ accordingto the invention is thus a protein which is capable of interacting witha double-stranded nucleic acid.

In a first embodiment of the method of the invention, the protein whichis used to block the renaturation of the denatured hairpin is a proteinwhich is capable of binding single-stranded nucleic acid. According tothis embodiment, the method of the invention thus relates to a methodfor the determination of the binding of a protein to a nucleic acidsequence, said method comprising the steps of:

-   -   denaturing a said hairpin molecule comprising the said sequence        by applying a physical force to the said molecule;    -   providing the said protein;    -   renaturing said hairpin molecule in the presence of the said        protein and    -   detecting a blockage of the renaturation of the hairpin.

Advantageously, the said method comprises the further step ofdetermining the position of the blockage.

In this embodiment, the invention relates to a method for thedetermination of the binding of single-stranded nucleic acid-bindingprotein to a nucleic acid sequence, said method comprising the steps of:

-   -   a) denaturing a hairpin obtained by the method described above,        said hairpin potentially comprising said sequence, by moving        away the surfaces;    -   b) measuring the distance Z_(high) between the two ends of the        denatured hairpin molecule obtained in step a);    -   c) contacting said protein with said denatured hairpin molecule        obtained in step a);    -   d) renaturing said hairpin molecule of step d) in the presence        of said protein; and    -   e) detecting a blockage of the renaturation of the hairpin        molecule; and    -   f) determining the position of said blockage with respect to one        end of the hairpin, said determination comprising the steps of:        -   measuring distance (z) between the two ends of the hairpin            molecule which are attached to a support,        -   comparing z and Z_(high), and        -   determining the position of the blockage;    -   g) determining the duration of said blockage,        wherein the binding of said single-stranded nucleic acid-binding        protein to said nucleic acid sequence is derived from the        position of said blockage and the duration of said blockage.

In another embodiment of the method of the invention, the protein bindsdouble-stranded nucleic acid. The inventors have shown that when adouble-stranded nucleic acid-binding protein is present, it is capableof binding the hybrid formed between a denatured hairpin and asingle-stranded nucleic acid molecule. This interaction between theprotein and the nucleic acid hybrid leads an alteration of the durationof the blockage. Most of the time, this interaction leads to anincreased blockage of the renaturation. For example, a primase willstabilize DNA oligos that would not otherwise have been sufficientlystable to block a hairpin re-hybridization for a time long enough to bedetected. Likewise, the binding of a DNA-polymerase to the 3′ end of asmall oligonucleotide used as a primer increases its stability.Alternatively, the duration of the blockage may also be reduced. Indeed,the present inventors have shown that the binding of some helicasestrigger a destabilization of the said hybrid, which is translated in ashorter blockage time.

According to this preferred embodiment, the method of the invention thuscomprises the steps of:

-   -   a) denaturing a hairpin molecule comprising a specific sequence        by applying a physical force to the said molecule;    -   b) providing the said protein and a single-stranded nucleic acid        molecule corresponding to the said nucleic acid sequence;    -   c) renaturing the said hairpin molecule in the presence of the        said protein and the said single-stranded nucleic acid molecule;        and    -   d) detecting a blockage of the renaturation of the hairpin.

Advantageously, the said method comprises the further step ofdetermining the position of the blockage.

In this embodiment, the invention relates to a method for thedetermination of the binding of double-stranded nucleic acid-bindingprotein to a nucleic acid sequence, said method comprising the steps of:

-   -   a) denaturing a hairpin obtained by the method described above,        said hairpin potentially comprising said sequence, by moving        away the surfaces;    -   b) measuring the distance Z_(high) between the two ends of the        denatured hairpin molecule obtained in step a);    -   c) hybridizing a single-stranded nucleic acid molecule with said        denatured hairpin molecule obtained in step a);    -   d) contacting said protein with said hybridised single-stranded        nucleic acid molecule/hairpin molecule of step c);    -   e) renaturing said hybridised single-stranded nucleic acid        molecule/hairpin molecule of step c) in the presence of said        protein; and    -   f) detecting a blockage of the renaturation of the hairpin        molecule; and    -   g) determining the position of said blockage with respect to one        end of the hairpin, said determination comprising the steps of:        -   measuring distance (z) between the two ends of the hairpin            molecule which are attached to a support,        -   comparing z and Z_(high), and        -   determining the position of the blockage;    -   h) determining the duration of said blockage,        wherein the binding of said double-stranded nucleic acid-binding        protein to said nucleic acid sequence is derived from the        position of said blockage and the duration of said blockage.

This embodiment is particularly advantageous because it allows for thedetermination of the binding of the said protein to the sequencecomprised within the double-stranded nucleic acid. It is possible tosequence directly the molecule bound by the said protein, withoutaltering the setup of the experiment, by just replacing the buffercontaining the protein and optionally a complementary single-strandednucleic acid, by a buffer suitable for sequencing according to themethod described above. The present method for determining the bindingof a protein to a sequence can thus be used to identify the binding siteof said protein. This method can notably be used for performing agenome-wide mapping of the binding sites of a specific nucleicacid-binding protein, such as e.g., a transcription factor. In thiscontext, it is particularly advantageous to use a library of hairpins,wherein said hairpin library represent a whole population of nucleicacids corresponding to the totality of the genome.

Another particular application of the method of the invention is in thedetection of epigenetic modifications. The present invention provides aneasy method for detecting epigenetic modifications of nucleic acids. By‘epigenetic modifications’, it is herein referred to modifications ofthe bases constituting a nucleic acid molecule which take place afterthe synthesis of said nucleic acid molecule. Such epigeneticmodifications include, inter alia, 4-methylcytosine (m4C),5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine(5fC) and 5-carboxylcytosine (5caC), as well as 6-methyladenosine (m6A)in DNA, and 5-hydroxymethyluracil (5hmU) and N6-methyladenosine (m6A) inRNA.

Likewise, the method of the invention allows the detection of modifiedbases resulting from nucleic acid damage, preferably DNA damage. DNAdamage occurs constantly because of chemicals (i.e. intercalatingagents), radiation and other mutagens. DNA base modifications resultingfrom these types of DNA damage are wide-spread and play important rolesin affecting physiological states and disease phenotypes. Examplesinclude 8-oxoguanine, 8-oxoadenine (oxidative damage; aging,Alzheimer's, Parkinson's), 1-methyladenine, 6-O-methylguanine(alkylation; gliomas and colorectal carcinomas), benzo[a]pyrene diolepoxide (BPDE), pyrimidine dimers (adduct formation; smoking, industrialchemical exposure, UV light exposure; lung and skin cancer), and5-hydroxycytosine, 5-hydroxyuracil, 5-hydroxymethyluracil, and thymineglycol (ionizing radiation damage; chronic inflammatory diseases,prostate, breast and colorectal cancer).

According to these embodiments, the presence of at least one modifiedbase in the sequence of interest contained in the hairpin of theinvention is identified by the detection of the binding of proteinrecognizing specifically said modified base (e.g., an antibody directedagainst said base) to said hairpin by the method described above.

These embodiments are particularly advantageous since all theoccurrences of a specific modification in a genome can thus beidentified and mapped accurately. In this context, it is particularlyadvantageous to use a library of hairpins, wherein said hairpin libraryrepresent a whole population of nucleic acids corresponding to thetotality of the genome.

Further embodiments and applications of the present method for thedetection of the binding of a protein to a specific nucleic acidsequence can be found in WO 2014/114687.

The practice of the invention employs, unless other otherwise indicated,conventional techniques or protein chemistry, molecular virology,microbiology, recombinant DNA technology, and pharmacology, which arewithin the skill of the art. Such techniques are explained fully in theliterature. (See Ausubel et al., Current Protocols in Molecular Biology,Eds., John Wiley & Sons, Inc. New York, 1995; Remington's PharmaceuticalSciences, 17th ed., Mack Publishing Co., Easton, Pa., 1985; and Sambrooket al., Molecular cloning: A laboratory manual 2nd edition, Cold SpringHarbor Laboratory Press—Cold Spring Harbor, N.Y., USA, 1989).

The examples below will enable other features and advantages of thepresent invention to be brought out.

LEGENDS OF THE FIGURES

FIG. 1: Structure of DNA hairpins used in magnetic tweezer analysis. Atypical DNA hairpin structure is shown. The bold sequence represents thedouble stranded DNA region of interest, and the various DNA linkersrequired for functionality are described. Linker 1 is a small DNA loopthat permanently attaches the 5′end of one strand of the ROI to the 3′end of the other strand. The structure will readily bind to astreptavidin-coated bead, by virtue of the Biotin moiety (shown as a reddot) synthesised on the end of linker 2. Finally, linker 3 allowsbinding to the flow cell surface coated in anti-digoxigenin antibodies,through the interaction with the digoxigenin located at its end (greendot).

FIG. 2: Principle of the stand invasion process. On the far left auniversal precursor hairpin-bead construct (HP1) is shown, which can beprepared in bulk and attached to the surface of the flow cell. It has adsDNA linker attached to the bead, ending with a ssDNA overhang ofsequence D-A. A second molecule consists of a dsDNA region with adigoxigenin-labelled tail allowing it to bind to the glass surface ofthe flow cell (dig represented by orange squares). The other end has assDNA region with a sequence C (of 12 nts) plus the 40 nt complementarysequence to A (A′). These two molecules (bead+linker and dig labelledlinker) can be pre-incubated so that they hybridise together via the Aand A′ sequences, forming pre-hairpin structure HP1. This construct willbe stable under normal conditions, but pulling on the magnetic bead witha force greater than a few pN will unzip the hybridised region anddissemble the structure. Next to this construct is displayed thestructure called hairpin precursor HP2. It consists of the (doublestranded) DNA to be studied (in grey) ligated to a loop at one end(shown on the right) and an adaptor at the other. The adaptor consistsof 2 oligos hybridised together via A and A′ sequences (identical tothose on the hairpin shown in the left panel). The adaptor also hasoverhanging single stranded regions of sequences D′ and C′ that arecomplementary to sequences C and D of HP1, respectively. When a libraryof these hairpins (HP2) is introduced to a flowcell containing aplurality of the structures shown in the left panel, they will hybridisethrough their flaps (C′ and D′) each forming a Holiday junction with 2nicks, called here HP3 (as shown in the middle right panel). When asmall force (˜5 pN) is applied to the bead, the Holiday junction rapidlymigrates, lengthening the molecule and leading to a stable hairpinconstruction (shown in the far right panel). The molecule shown in theright panel is essentially identical to that shown in FIG. 1; it can bezipped and unzipped as usual for such hairpin structures, and issuitable for all mapping and sequencing experiments. Note that althoughthere are 3 single stranded nicks in this molecule, the length ofcomplementary sequence (AD-A′D′ and CA′-C′A) is long enough to resistthe shearing force of the magnet (as opposed to the smaller length ofcomplementary sequence found on the uninvaded Halliday structure (heldtogether only by 12 bp D-D′ and C-C′).

FIG. 3: Alternative strand invasion process. This example is similar tothat shown in FIG. 1, with the exception that the invading hairpin hasonly a single flap (C′) with which to bind to proto-hairpin structureaffixed to the surface. The structure of the resulting strand-invadedhairpin is very similar to that in FIG. 2.

FIG. 4: Examples of fingerprints obtained with an oligonucleotideCGCCAC. A hairpin was generated with the 1.6 kb BsmBI fragment obtainedfrom pPS002 digestion. The force on the bead was gradually increaseduntil it reached a point where the molecule unziped. Reduction of theforce caused reziping of the molecule. In the absence of anyoligonucleotide, the closing was rapid (left panel). However, when theoligonucleotide was present in the flow cell and the complementarysequence of this oligonucleotide is on the hairpin, it blocked thereziping. This oligonucleotide had 3 binding positions on the hairpin,only one (at position 794 bp) is showing blockage due to the nature ofthe oligonucleotide. The experimental value obtained for this blockageon the particular bead was 784 bp. This oligonucleotide also blocked thereformation of the hairpin due to a blocking site located within thePS046 loop oligonucleotide, although there is a mismatch at the 5′ endbetween the oligonucleotide and its target sequence.

FIG. 5: Detection of the 5-methylcytosine modification. The same hairpincreated through FISI with the BsmBI digested fragment from pPS003 wastested against the 5-methylcytosine modification with antibodies (theclone 33D3 monoclonal antibody was used in this experiment and iscommercially available from various sources such as Merck Millipore orSigma-Aldrich). This hairpin was predicted to contain 2 potential Dcmmethylation sites at position 170 and 1046. Using the ssDNA blockage asa reference (the first one from the top), the experimental blockingposition were calculated to be at 135 bp and 1035 bp. Both theoligonucleotide and the antibody confirmed that the fragment of DNA wasreally originating from pPS003.

EXAMPLES Example 1

Preparation of HP1 Containing One Flap

The pPS001 vector (SEQ ID NO. 1) was used to clone a 1.5 kb KpnIfragment or a 500 bp SalI fragment from lambda genomic DNA to yieldpPS002 (SEQ ID NO. 2) or pPS003 (SEQ ID NO. 3), respectively.

To create the dsDNA linker between the bead and the precursor HP1,DreamTaq DNA polymerase was used according to the manufacturerspecifications. The oligonucleotides PS079 (SEQ ID NO. 6) and PS080 (SEQID NO. 7) were used at 500 nM concentration each and depending on thedesired length of the linker, either pPS001, pPS002 or pPS003 vectorswas used as template (creating a linker of either 236, 1724 or 737 basepairs, respectively).

The oligonucleotide PS080 contains a biotin at its 5′ end. Theoligonucleotide PS079 has a 12-carbon spacer (C12 spacer) that preventsthe DNA polymerase from “copying” the 5′ end of the vector, and leaves a5′, single stranded tail.

The PCR conditions were as follow:

$\begin{matrix}{\;{98{^\circ}\mspace{14mu}{C.\mspace{14mu}{for}}\mspace{14mu} 3\mspace{14mu}\min}} \\{\left. \begin{matrix}{58{^\circ}\mspace{14mu}{C.\mspace{14mu}{for}}\mspace{14mu} 20\mspace{14mu}\sec} \\{72{^\circ}\mspace{14mu}{C.\mspace{14mu}{for}}\mspace{14mu} 1\mspace{14mu}\min} \\{95{^\circ}\mspace{14mu}{C.\mspace{14mu}{for}}\mspace{14mu} 30\mspace{14mu}\sec}\end{matrix} \right\} 30X} \\{\;{72{^\circ}\mspace{14mu}{C.\mspace{14mu}{for}}\mspace{14mu} 5\mspace{14mu}\min}} \\{\;{{Hold}\mspace{14mu}{at}\mspace{11mu} 4{^\circ}\mspace{14mu}{C.\mspace{14mu}{for}}\mspace{14mu}{ever}}}\end{matrix}$

For the adapter between the HP1 and the surface, DreamTaq DNA polymerasewas used according to the manufacturer specification with theoligonucleotides PS103 (SEQ ID NO. 8) and PS104 (SEQ ID NO. 9) on thetemplate sequence pPS003. The same conditions as previously were used.

The resulting sequence is as follow (including the sequence added tocreate Bsal restriction site in green):

SEQ ID NO. 5: 5′ attcgatcgtGGTCTCAGAATcctggtggtgagcaatggtttcaaccatgtaccggatgtgttctgccatgcgctcctgaaactcaaCatcgtcatcaaacgcacgggtaatggattttttgctggccccgtggcgttgcaaatgatcgatgcatagcgattcaaacaggtgctggggcaggccTttttccatgtcgtctgccagttctgcctctttctcttcacgggcgagctgctggtagtgacgcgcccagctctgagcctcaagacGCTTGGAGACCagctagccat 3′

The resulting fragment was then digested with Bsal according to themanufacturer specification. On these overhangs, two different adaptersare then cloned as follows:

-   -   The first of these adapters is obtained by hybridizing        oligonucleotides PS101 (SEQ ID NO. 10) and PS070 (SEQ ID NO.        11). The resulting 5′ overhang of PS101 is complementary to one        of the fragment overhangs.    -   The second adapter is obtained by hybridizing oligonucleotides        PS101 and PS0102 (SEQ ID NO. 12). Both ends of PS102 extend        beyond PS101. One of these ends is complementary to the second        overhangs of the fragment.

Specifically, the oligonucleotides PS101 and PS070 were annealed inCutSmart buffer (NEB, 1×: 50 mM Potassium Acetate, 20 mM Tris-acetate,10 mM Magnesium Acetate, 100 μg/ml BSA, pH 7.9@25° C.) at aconcentration of 10 μm each. The solution was heated at 98° C. and letcool down to room temperature on the heat block. The same procedure wasperformed for the oligonucleotides PS101 and PS102.

Two different adapters were thus created, that can be ligated to theBsal digested PCR fragment directionally due to the presence of 2different, non-palindromic, sites using T4 DNA ligase from Enzymaticsaccording to manufacturer specification.

Once ligated, the oligonucleotide PS101 was extended using PS070 astemplate using Klenow exo-DNA polymerase from Enzymatics. The amount ofdTTP in the reaction mix was adjusted such that dig-dUTP could be used.There was a ratio of 40% dTTP for 60% of dig-dUTP.

After purification of the final fragment on agarose gel, the molarity ofboth fragments was calculated. The two fragments were then mixedtogether at equal molarity in CutSmart buffer. The 3′ end of theoligonucleotide PS102 is complementary to the 5′ end of theoligonucleotide PS079, after the C12 spacer.

Once annealed, the final HP1 molecule was serially diluted and bound onMyOne paramagnetic beads functionalized with streptavidin. Since thePS080 oligonucleotide contains a biotin, the molecule will bind to thebeads. The precursor HP1 was then loaded in the microfluidic chamber,the floor of the cell being functionalized with anti-digoxigeninantibodies. Due to the presence of digoxigenin on the second part ofHP1, the precursor HP1 binds to the floor of the flow cell.

Preparation of HP2

For the production of HP2, the BsmBI fragment from the vector pPS002(SEQ ID NO. 2) was obtained through digestion of the vector and purifiedfrom the gel. The resulting 1.6 kb fragment had 2 differentnon-palindromic ends.

The oligonucleotides PS108 (SEQ ID NO. 13) and PS109 (SEQ ID NO. 14)were mixed at equal concentration (10 μM) in CutSmart buffer and heatedat 98° C. Then, the tube was slowly cooled down to room temperature toallow the 2 oligonucleotides to anneal, thus creating an adapter withthe complementary overhang to the digested BsmBI fragment.

PS046 (SEQ ID NO. 15) is a self-annealing oligonucleotide, with a loopof 5 thymines and a 5′ overhang enabling cloning to a BsmBI digestedvector. PS046 was diluted to 10 μM, heated at 98° C. and rapidly cooleddown on ice, in order to promote the formation of a small hairpin-loopstructure which can be ligated to the DNA region of interest (formingHP2).

Once ligated to the BsmBI fragment, the resulting HP2 was purified ontoan agarose gel and eluted into 50 μl of water. 1 μl of this HP2 wasmixed with 100 μl of passivation buffer and loaded on the fluidic cellcontaining the HP1 attached to the surface. The magnet was brought closeto the sample such that the force reached around 5 pN. The sample wasleft like this for 30 minutes and the force was gradually increased to15 pN. If an ssDNA flap (C′) from HP2 has hybridized to thecomplementary sequence C on HP1, the Holliday junction would beresolved. In case there is no HP2 attached to HP1, the bead would flyaway. Once resolved, the hairpin can be interrogated with eitheroligonucleotides or any other binding molecules like antibodies orproteins.

Example 2

Preparation of HP1 Molecule Containing Two Flaps.

For this version, the hairpin of interest contains two ssDNA stretchesthat can bind on either side of the fork.

For the 2 flap strategy, the procedure is basically the same except thatthe oligonucleotides used are slightly different.

For the biotin-Space linker, there is no change. PS079 and PS080 wereused on pPS003 vector

For the dig linker, PS102 was replaced by the oligonucleotide PS115 (SEQID NO. 16). The latter was then annealed with PS101 to create theadapter to be ligated with the Bsal digested PCR fragment obtained withPS103-PS104. The second adapter, PS101-PS070 was unchanged. The dig-tailwas then synthesised as previously described.

Both fragments were purified on agarose gel and mixed at equal amount toform the HP1. Then, they were serially diluted and bound to MyOneparamagnetic beads coated with streptavidin.

For making the HP2, the PS107-PS108 adapter were replaced with theadapter composed of PS116 (SEQ ID NO. 17)-PS118 (SEQ ID NO. 18) to makethe two flap HP2. They were mixed in CutSmart buffer at 10 μM, heated at98° C. and slowly cooled to room temperature. They were finally ligatedas well as the loop PS046 to the BsmBI fragment from pPS002 vector.

The final fragment was purified on agarose gel and 1 μl of the resultingeluate was loaded inside the flow cell containing the HP1 precursor. Thesame procedure as previously was applied.

The invention claimed is:
 1. A method for preparing a hairpin nucleicacid comprising a sequence of interest, said method comprising the stepsof: (a) providing a nucleic acid HP1 , said nucleic acid HP1 comprising:a first end bound to a first surface; a single-stranded sequence Alinked to said first end, a single-stranded sequence A′ hybridized tosaid sequence A, wherein said sequence A and said sequence A′ are notcovalently linked; and form a double stranded region compromising thesequence A and the sequence A′; a single-stranded sequence C linked tosaid sequence A′; a second end linked to said sequence C, wherein saidsecond end is bound to a second surface; wherein one of the firstsurface and the second surface is a movable surface and the firstsurface and the second surface are different surfaces; b) providing atleast one nucleic acid HP2, said nucleic acid HP2 comprising: adouble-stranded region comprising the sequence of interest, a looplinked to a first end of said double-stranded region that links the twostrands of said double-stranded region, a single-stranded region havingthe sequence A linked to a first strand of a second end of saiddouble-stranded region, said single-stranded region having the sequenceA being linked to a single-stranded sequence C′, the sequence C′ beingcomplementary to the sequence C; a single-stranded polynucleotide havingthe sequence A′ linked to a second strand of the second end of saiddouble-stranded region wherein the sequence A hybridizes to the sequenceA′and form a double stranded region comprising the sequence A and thesequence A′; c) denaturing said nucleic acid HP1 from step a) in thepresence of said nucleic acid HP2 from step b) by moving one surface ofthe first surface and the second surface away from another surface ofthe first surface and the second surface by applying a tension of atleast 3 pN to the moveable surface, such that said double strandedregion comprising the sequence A and the sequence A′ of said nucleicacid HP1 are completely denatured; and d) obtaining said hairpin nucleicacid, wherein said hairpin nucleic acid is formed by hybridizing saidnucleic acid HP1 to said nucleic acid HP2 in the presence of thetension.
 2. The method of claim 1, wherein: said sequence A of saidnucleic acid HP1 is linked to a single-stranded sequence D, saidsequence D being located between said sequence A and said first surfaceand said sequence A linked to said first end bound to the first surfaceby said sequence D; and said sequence A′ of said nucleic acid HP2 islinked to a single-stranded region having sequence D′, wherein thesequence D′ is complementary to the sequence D of said nucleic acid HP1.3. The method of claim 2, wherein each of the sequence D and thesequence D′ comprises at least 10 nucleotides.
 4. The method of claim 2,wherein each of the sequence D and the sequence D′ comprises at least 12nucleotides.
 5. The method of claim 2, wherein each of the sequence Dand the sequence D′ comprises at least 13 nucleotides.
 6. The method ofclaim 1, wherein the tension of at least 3 pN is a tension of at least 4pN.
 7. The method of claim 1, wherein more than one molecule of saidnucleic acid HP1 is attached to one of the first surface and the secondsurface.
 8. The method of claim 1, wherein said at least one nucleicacid HP2 comprises a plurality of distinct nucleic acid molecules. 9.The method of claim 1, wherein each of the sequence A and the sequenceA′ comprises at least 30 nucleotides.
 10. The method of claim 1, whereineach of the sequence C and the sequence C′ comprises at least 10nucleotides.
 11. The method of claim 1, further comprising: e)completely denaturing the hairpin structure in the hairpin nucleic acidby moving one surface of the first surface and the second surface awayfrom another surface of the first surface and the second surface andobtaining a nucleic acid molecule without the hairpin structure; f)measuring the distance (Z_(high)) between the two ends of the nucleicacid molecule without the hairpin structure obtained in step e); g)hybridizing a single-stranded nucleic acid molecule with said nucleicacid molecule without the hairpin structure obtained in step e) andgenerating a complex; h) renaturing said hairpin structure of saidcomplex from step g); i) detecting a blockage of the renaturation ofsaid hairpin structure of said complex, wherein said blockage is causedby said single-stranded nucleic acid molecule of said complex; and j)determining the position of said blockage with respect to one end of thehairpin nucleic acid, said determination comprising the steps of:measuring distance (z) between the two ends of the hairpin nucleic acidwhich are attached to the first surface and the second surface duringthe period of said blockage, comparing z and Z_(high), and determiningthe position of the blockage with respect to one end of the hairpinnucleic acid.
 12. The method of claim 1, further comprising: e)completely denaturing the hairpin structure in the hairpin nucleic acidby moving one surface of the first surface and the second surface awayfrom another surface of the first surface and the second surface andobtaining a nucleic acid molecule without the hairpin structure; f)measuring the distance (Z_(high)) between the two ends of the nucleicacid molecule without the hairpin structure obtained in step e); g)hybridizing a single-stranded nucleic acid molecule with said nucleicacid molecule without the hairpin structure obtained in step e) andgenerating a complex; h) renaturing said hairpin structure of saidcomplex from step g); i) detecting a blockage of the renaturation ofsaid hairpin structure of said complex, wherein said blockage is causedby said single-stranded nucleic acid molecule of said complex; j)determining the position of said blockage with respect to one end of thehairpin nucleic acid, said determination comprising the steps of:measuring distance (z) between the two ends of the hairpin nucleic acidwhich are attached to the first surface and the second surface duringthe period of said blockage, comparing z and Z_(high), and determiningthe position of the blockage with respect to one end of the hairpinnucleic acid; and k) determining the duration of said blockage.
 13. Themethod of claim 1, further comprising: e) completely denaturing thehairpin structure in the hairpin nucleic acid by moving one surface ofthe first surface and the second surface away from another surface ofthe first surface and the second surface and obtaining a nucleic acidmolecule without the hairpin structure; f) measuring the distance(Z_(high)) between the two ends of the nucleic acid molecule without thehairpin structure obtained in step e); g) contacting a single-strandednucleic acid-binding protein with said nucleic acid molecule without thehairpin structure obtained in step e) and generating a complex; h)renaturing said hairpin structure of said complex from step g) in thepresence of said protein; i) detecting a blockage of the renaturation ofthe hairpin structure, wherein said blockage is caused by binding saidprotein to a single-stranded region of said nucleic acid moleculewithout the hairpin structure; j) determining the position of saidblockage with respect to one end of the hairpin nucleic acid, saiddetermination comprising the steps of: measuring distance (z) betweenthe two ends of the hairpin nucleic acid which are attached to the firstsurface and the second surface during the period of said blockage,comparing z and Z_(high), and determining the position of the blockagewith respect to one end of the hairpin nucleic acid; and k) determiningthe duration of said blockage.
 14. The method of claim 1, furthercomprising: e) completely denaturing the hairpin structure in thehairpin nucleic acid by moving one surface of the first surface and thesecond surface away from another surface of the first surface and thesecond surface and obtaining a nucleic acid molecule without the hairpinstructure; f) measuring the distance (Z_(high)) between the two ends ofthe nucleic acid molecule without the hairpin structure obtained in stepe); g) contacting a double-stranded nucleic acid-binding protein and asingle-stranded nucleic acid molecule with said nucleic acid moleculewithout the hairpin structure obtained in step e) and generating acomplex, wherein the single-stranded nucleic acid molecule hybridizes tosaid nucleic acid molecule without the hairpin structure; h) renaturingsaid hairpin structure of said complex from step g) in the presence ofsaid protein; i) detecting a blockage of the renaturation of the hairpinstructure, wherein said blockage is caused by binding said protein to adouble-stranded region in the complex formed by the single-strandednucleic acid molecule and a single stranded region generated by completedenaturation of the hairpin structure; j) determining the position ofsaid blockage with respect to one end of the hairpin nucleic acid, saiddetermination comprising the steps of: measuring distance (z) betweenthe two ends of the hairpin nucleic acid which are attached to the firstsurface and the second surface during the period of said blockage,comparing z and Z_(high), and determining the position of the blockagewith respect to one end of the hairpin nucleic acid; and k) determiningthe duration of said blockage.
 15. The method of claim 1, wherein thetension of at least 3 pN is a tension of at least 5 pN.
 16. The methodof claim 1, wherein the tension of at least 3 pN is a tension of atleast 6 pN.
 17. The method of claim 1, wherein each of the sequence Aand the sequence A′ comprises at least 35 nucleotides.
 18. The method ofclaim 1, wherein each of the sequence C and the sequence C′ comprises atleast 12 nucleotides.